PKR deficiency delays vascular aging via inhibiting GSDMD-mediated endothelial cell hyperactivation

Summary Vascular aging is an independent risk factor for cardiovascular diseases, but the regulatory mechanism is not clearly understood. In this study, we found that endothelial PKR activity is elevated in aging aorta tissues, which is accompanied with increased endothelial cell hyperactivation, IL-1β and HMGB1 release and vascular smooth muscle cell (VSMC) phenotype transforming. Global knockout of PKR exhibits significantly delayed vascular aging compared to wild-type mice at the same age. In vitro, using PKR siRNA or the cell hyperactivation inhibitor glycine or disulfiram can effectively inhibit H2O2 or palmitic acid-induced endothelial cell hyperactivation, IL-1β and HMGB1 release and co-cultured VSMC phenotype transforming. These results demonstrate that endothelial PKR activation induces GSDMD-mediated endothelial cell hyperactivation to release HMGB1 and IL-1β, which promotes the phenotype transforming of VSMC and subsequent accelerates the process of vascular aging. These discoveries will help to explore the new drug target to inhibit vascular aging.


INTRODUCTION
The population aging is an important economic and societal problem around the world. 1 Cardiovascular diseases (CVDs) such as hypertension, atherosclerosis, and stroke are the most common causes of death among elderly people worldwide. 2 According to the cardiovascular epidemiological statistics, the incidence of CVDs among people aged 25-44 is only 5.5%, but among people over 65, the incidence of CVDs is as high as 41%. 3 Moreover, more than 90% of patients with CVDs are over 40 years old. 4 Vascular aging, as one of the main physiological characteristics of aging, is regarded as an independent risk factor for CVDs. 5 With an increase in age, arterials gradually undergo a series of functional and structural alterations, including endothelial cell dysfunction, smooth muscle cell proliferation, and collagen deposition, which lead to an increase of vascular stiffness and decrease of blood flow. 3,6 However, our ability to slow or reverse the progression of vascular aging is limited because of our poor understanding of mechanisms that control this process. Therefore, it is of great significance to explore the molecular mechanism of vascular aging to prevent the process of age-related CVDs.
Double-stranded RNA-dependent protein kinase R (PKR) as a serine/threonine protein kinase can induce autophosphorylation at Thr 446 and Thr 451 sites by specifically binding to stimulus response elements, which plays an important role in regulating various signal pathways of innate immune diseases. 7 In the past, it was considered that PKR could only be activated by infectious agents, TLRs ligands, cytokines and other inherent immune-related factors, to regulate the activation of immune signal pathways and release of immune inflammatory factors. 8 In our recent studies, PKR has been revealed to be a key target in promoting endothelial cell senescence and pulmonary hypertension (PH) mediated endothelial injury. 9,10 In normal physiological conditions, the endothelium is a crucial regulator of vascular physiology and produces several substances to protect the layer of arteries. However, injured endothelial cells become the initial contributor to promote the development of cardiovascular diseases in a pathological phenotype. 11 We previously reported that PKR triggered IL-1b and HMGB1 release to induce PH development, although how endothelial PKR promotes IL-1b and HMGB1 release in vascular aging still need to be further investigated.
Gasdermin family member GSDMD has long been defined as the executor of pyroptosis, a novel programmed cell death. The N-terminal of GSDMD acts as the active fragment of GSDMD, mediates the pore-formation on cellular membrane to trigger cell membrane lysis-mediated cell death and inflammatory factors leak. 12 However, the latest studies have found that GSDMD mediated pore-formation can also induce inflammatory factors release in living macrophage cell, which is called cell hyperactivation. 13 In this cell state, activated GSDMD do not trigger cell death but only mediates the release of inflammatory factors and metal ion. Hence, we try to in-depth evaluate whether PKR mediated inflammatory factors release is because of mediating endothelial cell hyperactivation. Despite this, how endothelial PKR-mediated inflammatory factors release induces vascular smooth muscle cells (VSMCs) senescence is still unknown. As the main cell type within the vasculature, VSMCs are responsible for maintaining vascular homeostasis. It can present as contraction phenotype and secretory phenotype. 14 The phenotype transforming of VSMCs from contraction phenotype to secretory phenotype is a remarkable symbol of vascular aging. 2 In normal blood vessel, contraction phenotype VSMCs is the vast majority type of VSMCs. During the aging process, VSMCs gradually lose the contractile phenotype and acquire the proliferative and secretory phenotype, which eventually contributes to vascular degeneration and vascular remodeling via abnormal self-proliferation and promotes the vascular stiffness by the excessive deposition of collagen and decrease of elastin. 15 Therefore, we hypothesize that endothelial PKR-mediated inflammatory factors release can induce the phenotype transforming of VSMCs to induce vascular aging.
Taken together, we hypothesize that endothelial PKR induces GSDMD-mediated endothelial cell hyperactivation to release HMGB1 and IL-1b, which lead to the phenotype transforming of VSMCs and subsequent accelerates the process of vascular aging.

Activity of PKR is elevated in aging vascular endothelium
Our previous studies have revealed that PKR activation plays an important role in promoting human umbilical vein endothelial cells (HUVECs) senescence, but the role of PKR in regulating vascular aging is still unknown. To examine whether PKR contributes to vascular aging, qPCR and western blotting were performed in the thoracic aorta from 2-month-old mice (young mice) and 18-month-old mice (aging mice). Inconsistent with our previous studies that PKR expression and activity is elevated in aging HUVECs, 9 the elevated level of PKR activity also does not accompany an increase of PKR expression in aging thoracic aorta ( Figures 1A  and 1B). An increased PKR activation is mainly focused on the vascular intima ( Figure 1C). These results suggested that the elevated endothelial PKR activity may play important role in vascular aging.

Knockout of PKR delays vascular aging
To clarify the role of PKR in the vascular aging process, we compared the thoracic aorta morphological difference between 18-month-old PKR knockout mice with the same-month-old wildtype (WT) mice. As revealed by hematoxylin-eosin (H&E) and Masson staining, the thoracic aorta tissue from aging WT mice exhibited obvious vascular aging characteristics, as shown by increase of intima and media thickness (IMT) with increased collagen and decreased elastin content in media (Figures 2A-2C). However, aging PKR knockout mice appeared the alleviative thickening, collagen deposition and elastin decreasing in thoracic aorta tissue when compared to aging WT controls (Figures 2A-2C). At the same time, pro-senescence protein p53 and p16 as senescence-associated markers were also detected by immunohistochemical staining in vascular tissues. We found that pro-senescence protein p53 and p16 both strikingly increased in aging WT mice rather than aging PKR knockout mice ( Figures 2D and 2E). In addition, vascular aging is always accompanied with the decline of capillary density and blood flow. 16 Hence, blood flow intensity was evaluated in the hind limbs of mice by laser Doppler. Compared with young WT mice, the significantly decreased capillary density and blood flow in the hind limbs was shown inold WT mice ( Figures 2F and  2G). However, slightly decreased capillary density and blood flow of old PKR knockout mice when compared to young PKR knockout mice ( Figures 2F and 2G). These results suggest that PKR knockout can prevent vascular aging process and protect aging-induced vascular dysfunction.

Knockout of PKR inhibits the transforming phenotype of VSMC in aging vascular
As a prominent phenotype of aging aortas, the contractile phenotype of VSMCs was gradually replaced by the synthetic phenotype of VSMCs and subsequently caused the aortic wall thickening and stiffening in aging mammals. 17 To clarify the underlying mechanisms of whether PKR induced vascular aging by promoting the transforming phenotype of arterial VSMCs, western blotting showed markedly decreased expression of a-smooth muscle actin (a-SMA), smooth muscle 22 alpha (SM22a), caldesmon and calponin (contractile iScience Article phenotype markers) with increased level of thrombospondin and osteopontin (synthetic phenotype markers) in aorta of aging WT mice when compared with young WT mice ( Figure 3A). However, the aortas from aging PKR knockout mice did not show an obvious switch of synthetic phenotype to contractile phenotype in VSMCs. We also confirmed this phenomenon using immunohistochemical staining to detect the level of a-SMA, SM22a (contractile phenotype markers) and osteopontin (synthetic phenotype markers) ( Figure 3B). These results suggested that PKR promotes the transforming phenotype of VSMCs to induce vascular aging.
Interfering PKR reduces the release of IL-1b and HMGB1 We next investigated the mechanisms about how endothelial PKR promotes VSMCs phenotype transformation. Because inflammatory cytokines released from endothelial cells are major drivers of vascular aging, 18 we measured the cytokine levels in the plasma using ELISA. In previous studies, PKR has been reported to mediate the release of inflammatory cytokines including IL-1b and HMGB1, both of which have been verified to contribute to the process of aging. 19,20 We found that the IL-1b and HMGB1 concentration increased in the aorta of aging WT mice ( Figure 4A). Notably, their levels in aging PKR knockout mice aorta are not obviously altered. However, we still need to further verify whether PKR from senescence endothelial iScience Article cell induced the increased IL-1b and HMGB1 release. We suppressed PKR expression using small interfering RNA (siRNA) and demonstrated that PKR silence diminished pro-senescence stimulus H 2 O 2 mediated IL-1b and HMGB1 release in HUVECs ( Figure 4B).

Inhibiting PKR decreases GSDMD-mediated endothelial cell hyperactivation to prevent inflammatory factors release
Previous studies have reported that PKR promotes IL-1b and HMGB1 release in pyroptotic cell death. Hence, we detected whether PKR promotes pyroptosis in aging endothelial cells. Endothelial cells were stimulated with H 2 O 2 to induce senescence phenotype and PKR activation ( Figures 5A and 5B). However, unlike pyroptotic cell with GSDMD N-terminal (N-GSDMD) fragment formation and significant LDH release, endothelial cells with H 2 O 2 stimulation induce GSDMD N-terminal (N-GSDMD) fragment formation without LDH release ( Figures 5B and 5C). The HUVECs morphology was observed by a scanning  Figure 5D). In addition, similar results were demonstrated in aging vascular tissues. Although increased N-GSDMD was found in aging vascular tissues ( Figure 5E), but tunnel staining did not demonstrate increased cell death in aging PKR knockout mice of vascular endothelium ( Figure 5F). Despite this, knockdown of PKR effectively inhibited GSDMD N-terminal (N-GSDMD) fragment formation and pore-formation in HUVECs (Figures 5D and 5E). Taken together with the above results, we suggested that senescent endothelial cells are accompanied by cell hyperactivation rather than cell pyroptosis. Lacking PKR can block the development of cell hyperactivation in senescent endothelial cells.
To further confirm that GSDMD-mediated endothelial cell hyperactivation promotes the release of inflammatory factors, osmoprotectant glycine, known as an inhibitor of hyperactivation, was used to prevent membrane rupture and pyroptosis-induced cell lysis (Fink and Cookson, 2006). Compared with H 2 O 2treated group, glycine treatment did not decrease activation of GSDMD ( Figure 6A), but glycine greatly reduced the release of IL-1b and HMGB1 ( Figure 6B). In addition, an FDA-approved GSDMD pore formation inhibitor disulfiram was used to prevent H 2 O 2 -induced membrane rupture. 21 Similar to glycine, disulfiram failed to inhibit H 2 O 2 -induced cleavage of GSDMD but hindered the release of IL-1b and HMGB1 ( Figures 6C and 6D). These results indicate that PKR promotes GSDMD-mediated endothelial cell hyperactivation and subsequent inflammatory factors release.

Silencing endothelial PKR inhibits the phenotypic transformation of vascular smooth muscle
We further co-cultured HUVECs with HASMCs to investigate whether activated PKR-induced endothelial hyperactivation can trigger VSMCs phenotypic transformation ( Figure 7A). The phenotypic  Figure 7B). Moreover, performing glycine or disulfiram in H 2 O 2 -stimulated HUVECs also can inhibit the phenotypic transforming of co-cultured HASMCs ( Figures 7C and 7D).

Silencing endothelial PKR inhibits palmitic acid-induced endothelial cell hyperactivation and phenotypic transformation of vascular smooth muscle
Elevated palmitate acid (PA) level is an independent risk factor of cardiovascular diseases by promoting vascular endothelial cell senescence. 9 However, the role of PA on inducing endothelial cell hyperactivation and phenotypic transformation of vascular smooth muscle is still unknown. Similar to H 2 O 2 stimulation, PA also triggered endothelial cell hyperactivation and phenotypic transformation of co-cultured vascular smooth muscle by shearing GSDMD to N-GSDMD without LDH release and decreasing levels of a-SMA, SM22a, calponin and caldesmon with increasing level of thrombospondin and osteopontin (Figures 8A-8C). Knockdown of PKR by siRNA also inhibited PA-induced endothelial cell hyperactivation and phenotypic transformation of co-cultured vascular smooth muscle (Figures 8A-8C). Combined with above results, we suggested that silencing endothelial PKR inhibits the phenotypic transformation of vascular smooth muscle via blocking GSDMD-mediated endothelial cell hyperactivation, which may eventually repress the process of vascular aging. Previous studies proved that PKR is associated with neuroaging-related diseases, including Alzheimer's disease and neurodegenerative diseases. Our study further found that PKR also can promote vascular aging. Based on the results that the activity of PKR is especially increased in vascular endothelium and that global knockout of PKR sufficiently delays the vascular aging, we speculated that endothelial PKR is a key target of vascular aging. Aging endothelium is easily attacked by cytokines, inflammatory, metabolic products, and shearing force, all of which are also activators of PKR phosphorylation. PKR, as a crucial stress-responsive kinase, can be activated by virus, bacteria, cytokines, inflammatory factors, and metabolic products, even including oxidative stress and metabolic stress. 22 Our results may suggest that endothelial PKR can be activated by a variety of stress factors, which is the initial link to induce vascular aging.
However, we cannot totally exclude that PKR in blood cells also contributes to the progression of vascular aging. In previous studies, innate immune cells also associated the development of vascular aging. Abnormal monocyte-macrophages, neutrophils, T cells and dendritic cells are prominent in aging vascular remodeling. 23 Because PKR has long been verified to promote inflammatory factors release in immune cells, 20 whether the pro-inflammatory role of PKR in immune cells also promotes the development of vascular aging still requires further study.
Our results supported that PKR promotes GSDMD activation in aging vessels. However, unlike previous studies that reported that activated GSDMD mediates the development of pyroptosis, we found that PKR induces GSDMD-mediated cell hyperactivation in aging endothelial cells. In pyroptotic cells, GSDMD triggers plasma membrane rupture (PMR) and cell death, which is accompanied by the leakage of IL-1, HMGB1, other cytokines, and LDH. However, GSDMD mediated the secretion of IL-1 and other cytokines without LDH in living hyperactivated cells. We speculated that the reason why PKR induces hyperactivation but not pyroptosis in endothelial cells is because PKR cannot further spur GSDMD-dependent nerve injury-induced protein 1 (NINJ1) activation. 24 NINJ1, as a membrane protein, can be activated by caspase-11/GSDMD signaling and then lead to PMR, which causes pyroptotic cell death. 25 However, we speculated that overactivated PKR fails to mimic caspase11/GSDMD signaling mediated NINJ1 activation in aging endothelial cells, suggesting that endothelial inflammatory factors release rather than cell death is more important in promoting vascular aging.
In addition, previous studies reported that performing membrane rupture inhibitor glycine to pharmacologically inhibit GSDMD mediated pore formation can artificially switch LPS plus Nigericin mediated macrophage pyroptosis to cell hyperactivation. 13 Subsequent studies demonstrated that hyperactivated macrophages can secrete fibrin to promote disseminated intravascular coagulation (DIC) in endotoxemic mice. 26 Our results confirm that in endothelial cells, PKR/GSDMD pathway can specifically induce the In summary, the study confirmed that PKR has an essential role in the process of vascular aging. Given the global knockout of PKR effectively delaying vascular aging, targeting PKR may be of therapeutic benefit to

Limitation of the study
We demonstrated that endothelial PKR plays an important role in promoting vascular aging by PKR-global knockout mice and PKR siRNA in cells. However, this result should be further confirmed by specific PKR endothelial knockout mice and PKR knockout endothelial cells. Because PKR from innate immune cells iScience Article also promotes the release of HMGB1 and IL-1b which may mediate phenotypic transformation of vascular smooth muscle, we admitted that using HAoECs but not HUVECs is more suited to explain the mechanism of this study. In addition, the detection of PKR activity in clinical vascular samples is lacking, and the provascular aging role of PKR in clinical patients still requires further study.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: